We continue with our endeavor aimed at developing a conjugate vaccine for cholera from synthetic fragments of the O-PS of Vibrio cholerae O:1. A potent vaccine for the disease is not available. This work is important from the point of view of public health and, because of the involvement of our military in protecting US interests in developing third world countries, the availability of a vaccine for cholera has become of national interest. Cholera is a serious enteric disease caused mainly by two strains of Vibrio cholerae O:1, Ogawa and Inaba. The work towards a potent conjugate vaccine for cholera involves synthesis of oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugation of these antigens to suitable carriers, and serologic evaluation of the immunogenicity of the resulting neoglycoconjugates. The requisite oligosaccharides consist of 1?2-alpha-linked D-perosamine (4,6-dideoxy-4-amino-D-mannose) whose amino group is acylated with 3-deoxy-L-glycero-tetronic acid. Haptens required for conjugation result from multi step, sophisticated chemical syntheses, which is a very laborious process. Our original protocol for the synthesis involved assembly of the required oligosaccharide from intermediates lacking both the 3-deoxy-L-glycero-tetronic acid side chain and the spacer molecule. When the targeted oligosaccharide was assembled, the azido groups present were reduced to amino groups and the resulting amines were N-acylated with a suitable derivative of 3-deoxy-L-glycero-tetronic acid. Subsequently, the spacer molecule was attached to make the hapten amenable for chemical linking to a carrier. We later improved this protocol, to minimize the number of chemical modifications of the assembled oligosaccharide, and applied the new approach to the synthesis of tetra- and hexasaccharide fragments of the O-PS of Vibrio cholerae O:1, serotype Inaba. The new strategy is based on the use of glycosyl donors and glycosyl acceptors having the N-acyl side chain already in place. Also, the glycosyl acceptor is the N-3-deoxy-L-glycero-tetronylated perosamine glycoside of the spacer molecule (methyl 6-hydroxyhexanoate). Thus, when the requisite oligosaccharide is assembled the only chemical manipulation that needs to be carried out to obtain material suitable for conjugation is removal of protecting groups. Prompted by the findings of our previous work with constructs prepared from the Ogawa hexasaccharide and bovine serum albumin (BSA), which showed that the neoglycoconjugate is a potent immunogen with protective capacity for cholera, the objective of our work during this period was to find minimum size of the oligosaccharide that is sufficient to confer immunity. For this purpose, we have prepared a series of neoglycoconjugates from BSA and mono-, di-, tri-, tetra- and the pentasaccharides that mimic the terminal epitopes of the O-PS of Vibrio cholerae O:1, serotype Ogawa with varying carbohydrate density on the surface of the carrier. Monitoring of the conjugation by Surface Enhanced Laser Desrption Ionization- Time-of-Flight (MALDI-TOF) mass spectrometry enabled us to prepare conjugates in a predictable way. Constructs with predetermined carbohydrate?protein ratios (5, 10, and 15) were obtained. Within this work, we have obtained important information about the kinetics of the conjugation, and have also developed a protocol enabling preparation of a series of neoglycocnjugates with predetermined carbohydrate?protein ratios in a one-pot reaction. This ability is important in synthetic vaccine development as it greatly increases the efficiency of conjugation and our overall productivity. In collaboration with The Department of Bacteriology, Dartmouth Medical School, these materials were used to immunize small laboratory animals. The serological evaluation of the resulting antibody pools is currently under investigation.